Four serotypes of dengue virus (DENV1-4) circulate globally, causing more human illness than any other arthropod-borne virus. DENV infection results in dengue fever, an acute febrile illness, or the more severe, life- threatening dengue hemorrhagic fever/dengue shock syndrome. The greatest risk factor for severe dengue is a previous infection with a different DENV serotype. A dominant theory for why sequential heterotypic infections or transfer of maternal anti-DENV antibodies (Abs) to infants increases risk of severe disease is antibody- dependent enhancement (ADE), whereby anti-DENV Abs from a previous infection exacerbate a new infection of target Fc3 receptor-bearing cells, leading to inappropriate activation of macrophages and T cells and a cytokine storm that results in vascular leak. Due to recent advances in our mouse model for DENV infection and disease, in which we can model both ADE and Ab-mediated protection, we can now address critical questions about the role of serotype-, domain-, and epitope-specific Ab responses in protection or enhancement of DENV infection in vivo. The ability to compare Ab profiles generated using new-generation in vitro neutralization and enhancement assays with results obtained in our mouse model allows us to directly address the issue of in vitro correlates of in vivo outcome, a central tenet in dengue vaccine development. We are approaching these questions using complementary approaches by manipulating both polyclonal and monoclonal Abs and the virus itself. In Aim 1, we will evaluate the neutralizing and enhancing capacity of homotypic versus heterotypic Abs in serum as well as the role of domain-specific Abs by depleting mouse serum of Abs targeting different domains of the envelope protein. The role of specific epitopes will be addressed using a large panel of mouse and human anti-DENV mAbs. In Aim 2, ablation of particular epitopes will be evaluated using reporter viral particles as well as infectious clones. Importantly, the tools and methodologies developed in this grant will be used to evaluate the repertoire of anti- DENV Abs in human infections in our long-term prospective cohort and hospital-based studies in Nicaragua in separate work. Equally important for dengue vaccine development is improving our understanding of the memory immune response to DENV, and we will determine which B cell epitopes, as well as which memory T and B cell subsets, play a significant role in protection and/or enhancement in secondary DENV infection. In Aim 3, we will use complementary approaches of depletion, adoptive transfer, and immunization to define the role of memory T and B cell subsets and the contribution of B cell epitopes identified in Aims 1 and 2 in the context of a memory immune response. Thus, it is finally possible to dissect the mechanism of both neutralization/protection and enhancement of DENV infection and disease in a small animal model, which has direct implications for development of a safe tetravalent dengue vaccine.